This proposal will investigate mechanisms of progression from androgen-dependent to androgen-independent prostate cancer (AIPC). Our hypothesis is that one mechanism for AIPC development is mutations in the androgen receptor (AR) that widen its ligand binding specificity, and allow non-androgens to bind to and activate it, thus stimulating prostate cancer (PCa) growth. This sub-group of AIPC is therefore dependent on the AR and we refer to this pathway as the promiscuous AR. Examples include the ARs in LNCaP cells (mutation T877A), and MDA PCa cells (mutations T877A and L701H). Although often referred to as androgen-dependent, these cells have promiscuous ARs that are androgen-responsive but not dependent on androgens since flutamide and other steroids can stimulate their growth. The grant has 3 Specific Aims. Aim I will further evaluate MDA PCa 2a and 2b cells that we have recently described as a cause of AIPC due to a promiscuous AR that responds to corticosteroids (ARccr). We will further characterize the steroids that bind to ARccr and develop treatment strategies to block or suppress the ARccr. We will investigate the frequency of the L701H mutation and other AR mutations that cause the promiscuous AR phenotype in metastatic specimens. Finally, we will investigate the pattern of gene regulation by androgens and corticosteroids via the ARccr using cDNA microarray technology. In Specific Aim II we will study a novel treatment strategy for AR-dependent AIPC. We hypothesize that molecules that can selectively down-regulate AR (SARDs) will be therapeutically useful in treating AR-dependent AIPCs. We have identified a number of hormones and ligands with this activity. In Aim II we will further identify, characterize and elucidate the mechanism of action of SARDs as well as demonstrate their ability to diminish growth stimulation of AR- dependent AIPC cells. In Specific Aim III we will employ two in vivo models to investigate the treatment strategies developed in Aims I and II. Model 1 will employ classic xenografts in nude mice. Model 2 is a new approach using luciferase-transfected PCa cell xenografts that can be observed in live mice with a charge coupled device camera. The bioluminescent model will be a major improvement over current xenograft approaches providing repeatable, quantitative imaging of prostate cancer progression that can detect microscopic metastases. In conclusion, we propose in vitro and in vivo experiments to investigate the mechanism of transition of PCa from androgen-dependent to AIPC, focusing on mutations in the AR and treatment strategies to prevent the growth of AR-dependent PCa.